This proposal is designed to characterize the cell systems which contain motilin in several mammals including many be immunocytochemistry, nd to compare these results obtained by chemical analysis. The initial studies will further explore preliminary evidence that there are different forms of motilin in cells in different species and possibly in different organs in the same species. Antisera directed to different parts of motilin will be employed. Synthetic fragments of the molecule will be used for more specific antisera and for absorption tests with antisera including those presently in use. A major focus of this proposal concerns further definition of the motilin-containing Purkinje system of the cerebellum. At present motilin is the only peptide which is found in significant concentrations in cerebellum (25% of brain motilin is in cerebellum in rat). Since only Purkinje cells and their processes react to antiserum to motilin in cerebellum and none in brainstem, it seems likely that the heavy innervation observed in brainstem (particularly motor system neurons) nuclei represent direct innervations from cerebellum. The studies proposed using the antiserum to motilin as a new marker combined with lesion and tracing methods in rats will result in new concepts concerning direct efferent connections from cerebelum to brainstem (only direct connections known are to lateral vestibular mucleus which is also seen with anti-motilin). The relationship between GABA (GAD)-containing and motilin-reactive Purkinje cells will be investigated in mouse by immunocytochemistry and receptor autoradiography. Preliminary data suggests that these are different neurons. Immunoelectron microscopy will be employed to study possible recurrent collaterals on motilin cells and between motilin and GAD cells. The regional organization of motilin-reactive Purkinje neurons (more lateral than medial) will be investigated in detail in normal human and experimental animal cerebellum and in preparation for long term goals to study the development and pathological conditions involving the cerebellum. Such studies will be initiated in this proposal in rats regarding the toxic effects of phentoin and hypothyroidism on Purkinje cells.